It is well known that the genetic information of all cells is stored in deoxyribonucleic acid (DNA) in the chromosomal material of organisms. The unit of genetic function, i.e., the locus on the chromosome related to a specific hereditary trait, is called a gene.
Recombinant DNA technology involves the transfer of genetic material (genes, or DNA fragments) from one organism into a second organism, by means of a transfer component designated a "vector", producing a combination of genetic material. The second organism (which contains the transferred genetic material) is designated a recombinant component. The recombinant component is then inserted into bacterial and animal cells for propagation of the combined genes contained in the recombinant component. The cell into which the recombinant component is inserted is designated a host cell.
One type of vector comprises bacteriophages, which are viruses that infect bacteria. A typical bacteriophage consists of nucleic acid enclosed in a protein coat.
Using recombinant DNA technology, genetic modification can be accomplished as follows. Specific DNA fragments from bacteriophages and DNA from a bacteria are "isolated", e.g., by treatment with appropriate restriction enzymes which act as "chemical scalpels" to split DNA molecules into specific fragments which usually contain from less than 1 to 10 genes each, or by other well known techniques. A DNA fragment for the desired genetic characteristic, i.e., "foreign DNA" from the bacteria source is then inserted into the DNA bacteriophage vector. By treatment with DNA ligase the DNA fragment is inserted into the bacteriophage DNA vector and a recombinant bacteriophage DNA molecule is formed. The recombinant bacteriophage contains the genes of the bacteriophage plus the new genes (foreign DNA) from the inserted fragment. This recombinant bacteriophage can be introduced into a host bacterium thereby "cloning" the foreign DNA into the host. The new genes are propagated and become a part of the genetic machinery of the bacterium host. The bacterium host thus acquires the genetic traits contributed by the new genes and is capable of "expressing" these traits.
In recombinant technology work, a formidable task has been encountered in being able to "screen" the transformed cells and select the cells which have acquired the desired genetic trait, i.e. the viable transformants (hosts). In certain bacteria strains, a means for "primary" selection exists, e.g., if the transformed bacteria is resistant to a certain antibiotic the bacteria can be cultured in the presence of such antibiotic and the cells which survive can be selected as viable transformants. This process, involving genes which are vital to the survival of the bacteria, is designated primary selection. In contrast, genetic traits which are not vital to the survival of the bacteria, e.g., production of extracellular enzymes such as .alpha.-amylase, proteases, cellulases and hemicellulases cannot be selected on the basis of primary selection. There is thus a need for a recombinant technology method which is applicable to cloning both a gene for which primary selection does not exist, in addition to cloning a gene for which primary selection does exist.
The Bacillus genus contains approximately 48 species; virtually all of the species secrete a variety of soluble extracellular enzymes, under varying parental habitats. In addition, as discussed hereinafter, Bacillus microorganism also synthesize intracellular enzymes and antibiotics. Utilization of Bacillus microorganisms has reached commercial importance in such diverse fields as medicine and brewing. Further commercial utilization of Bacillus can be provided by the use of recombinant technology to allow the insertion of a variety of Bacillus genes encoding desired genetic traits into different Bacillus microorganisms such as B. subtilis.
It is known that genes encoding or regulating .alpha.-amylase in a Bacillus strain can be introduced into B. subtilis if the two strains are sufficiently closely related, i.e., if there is extensive genetic homology between the two strains. This is referred to as homologous cloning. For example, J. Bacteriol 120: 1144-1150 (1974) describes the introduction of DNA from B. subtilis var amylosaccharitus having exceptionally high .alpha.-amylase activity into a genetically similar (homologous) microorganism B. subtilis Marburg having relatively low .alpha.-amylase activity. The transformed organisms produced acquired high .alpha.-amylase activity.
However, most Bacillus are not sufficiently related to B. subtilis, i.e, are not sufficiently homologous, to permit the DNA obtained from one Bacillus subtilis strain to be efficiently introduced into a different Bacillus. J. Bacteriol 111: 705-716 (1972).
The literature (Kawamura, et. al.; Gene 5: 87-91 (1979)] describes a recombinant DNA technique which also involves insertion of DNA into a bacteriophage vector. Kawamura et. al. discloses isolation of chromosomal DNA fragments from a defective B. subtilis bacteriophage and insertion of the isolated DNA into another bacteriophage to produce a recombinant bacteriophage. The recombinant bacteriophage was then used in transformation of a B. subtilis microorganism and transformants were selected by conventional techniques. Because the chromosomal DNA was obtained from a defective B. subtilis bacteriophage, insertion of the DNA into a B. subtilis microorganism involved a homologous transformation. None of the prior art references discussed above discloses a method for introducing foreign DNA into a B. subtilis bacteriophage to produce a recombinant bacteriophage that can be used in heterologous cloning of genes.